A gene is a very important substance which expresses the traits of a living organism through transcription and translation and conveys the traits to its offspring. It is composed of a base sequence having A (adenine), C (cytosine), G (guanine) and T (thymine) as their base units. The base sequence of a gene determines the traits of a living organism. Analysis of the base sequence and nucleotide polymorphism of genes is very important in conducting researches on biological information. For example, nucleotide polymorphism analysis makes it possible to diagnose heredity related diseases and prescribe personalized medicine according to genetic traits. Also, the analysis allows to diagnose infection viruses and distinguish drug resistant bacteria. In addition, the analysis can be broadly applied to various fields such as species distinction of living organism and forensic medicine, etc.
Particularly, in the field of treatment of cancer, which has been the number one cause of death in Korea since 1983, various researches on genomic abnormalities have been underway since it was found that genomic abnormalities such as mutations of oncogenes, mutations or deregulations of tumour suppressor genes, and chromosomal abnormalities, etc. are directly involved in the occurrence of cancer and prognosis determination of drugs (Fearson E R et al., Cell., 1990, 61:759; K. W Kinzler et al., Cell., 1996, 87:159; Manuel Serrano et al., Cell., 1997, 88:593; Amado R G et al., J. Clin. Oncol., 2008, 26:1626; Raponi M et al., Curr Opin. Pharmacol., 2008, 8:413; Siena S et al., J. Natl. Cancer Inst., 2009, 101:1308).
For this reason, detection of mutations with clinical significance is very important, and accordingly a wide variety of detection methods which vary depending on the analysis purpose or genotype are continuously being reported (Taylor C F, Taylor G R. Methods Mol. Med., 92:9, 2004). Particularly, in somatic mutations, mutant genes exist at a very low frequency of about 0.1˜100 bases per megabase of wild type genes depending on the type of tumour. Also, the number of mutant cancer cells existing in analysis samples is considerably small compared to the number of normal cells, and thus it is very difficult to detect them and accordingly an advanced detection technique is required for the detection (Chung et al., Clin Endocrinol., 65:660, 2006; Trovisco et al., J Pathol., 202:247, 2004).
Representative methods for detecting a minute amount of mutation as in the above include various analysis methods based on real-time PCR technology, such as allele specific PCR method specifically amplifying mutants by using a mutant specific primer in order to selectively increase a small amount of mutant genes (Rhodes et al., Diagn mol pathol., 6:49, 1997), scorpion real-time allele specific PCR method (DxS' scorpions and ARMS) (Mark et al., Journal of Thoracic Oncology, 4:1466, 2009), CAST PCR method detecting amplification products excluding the position where mutation occurred by using Taqman probe after inhibiting amplification of wild type genes and selectively amplifying only mutant genes by using allele specific primer technology and minor groove binder (MGB)-probe (Didelot A et al., Exp Mol Pathol., 92:275, 2012), cold-PCR method increasing sensitivity of mutants by using critical denaturation temperature (Tc) (Zuo et al., Modern Pathol., 22:1023, 2009), etc. Such technologies can be applied easily and quickly to various diagnoses, and are good technology for diagnosing and analyzing mutation of cancer related genes (Bernard et al., Clinical Chemistry, 48:1178, 2002).
However, in case of the above methods, it is difficult to design experiments for reasons such as that a primer amplifying only mutants should be designed, and the critical denaturation temperature should be precisely maintained, etc. Also, in the method using an allele specific primer, false positive results may be obtained if mispriming occurs. Also, Taqman and scorpions probe methods, currently most widely used, have problems that they are not capable of conducting simultaneous multiple analysis using melting curve analysis of a probe, and thus that the number of genes detectable in one tube depends on the number of fluorescences detectable by the real-time PCR apparatus.
Recently, various molecular diagnosis technologies have been developed for detecting somatic mutation through real-time PCR technology. However, in the aspect of usefulness, they did not achieve a remarkable development, and it is necessary to develop a technology with high sensitivity and specificity which is capable of simultaneous multiple quantitative analysis in a short period of time.
Peptide nucleic acid (PNA) was reported in 1991 by Nielsen as a nucleic acid analogue having N-(2-aminoethyl)glycinamide as its backbone (Nielsen P E et al., Science, 254(5037):1497, 1991). The PNA backbone is electrically neutral, and thus has higher specificity and selectivity than DNA probe with respect to target nucleic acids having a complementary base sequence. Also, it is possible, by introducing a specific functional group at the alpha (a), gamma (y) position or linker part of the backbone, to freely adjust physical properties of PNA, such as cell penetration and its melting temperature from the target nucleic acid (Englund E A et al., Angew. Chem. Int. Ed. Engl., 46:1414, 2007; Stefano Sforza et al., Eur J. Org. Chem., 16:2905, 2000; Roberto Corradini et al., Curr Top. Med. Chem., 11:1535. 2011). Further, it has an advantage of not being decomposed by nuclease or protease, and thus is very useful in molecular diagnosis methods using a probe (Egholm et al., Nature, 365:556, 1993; Nielsen et al., Bioconjugate Chem., 5:3, 1994; Demidov, et al., Biochem. Pharmacol., 48:1310, 1994). Using these advantages of PNA, the PCR clamping technology was developed in 1993 (Henrik Orum et al., Nucleic Acids Res., 21:5332, 1993). This technology inhibits PCR amplification of a gene that should not be amplified by binding a PNA probe to the gene. When a PNA probe complementary to a wild type gene is used, the amplification of the wild type gene is inhibited during PCR reaction, thus making it possible to quickly and accurately detect mutants, which are present in a minute amount when compared to the wild type gene.
Currently, various techniques using the PNA clamping technology are being reported. Hereinafter, the characteristics of methods for selectively amplifying genes by using the PNA clamping technique will be briefly described.
PNA-LNA clamp method (US Patent Publication No. 2013-0005589; Yoshiaki Nagai et al. Cancer Res., 65(16):7276, 2005) is a method for selective amplification and selective detection designed such that a PNA clamping probe having a wild-type gene sequence and an LNA taqman probe for detection having a mutant gene sequence competitively hybridize with the target site. However, this method uses the taqman probe method where a DNA polymerase having 5′→3′ exonuclease activity degrades a mutation probe labeled with a fluorescent substance and a quencher to allow fluorescence. Thus, according to this method, the melting temperature (Tm) value of the probe cannot be analyzed, and it is not possible to detect multiple targets with one fluorescence.
PNA hyb probe (PNA as both PCR clamp and sensor probe; US Patent Publication No. 2008-0176226) is a technology designed to allow a PNA probe to conduct clamping and detecting simultaneously by changing the donor probe in Roche's conventional hyb probe system to a PNA probe. However, since the technology still has a limitation that it must comprise an anchor probe, it is difficult to design the probe, and the use of a long anchor probe disables simultaneous detection of multiple mutations at adjacent positions. Also, the use of one PNA probe conducting clamping and detecting simultaneously makes difficult an analysis in determination of multiple mutant genotypes within the same codon through the melting curve analysis of the PNA probe because the melting temperatures of the PNA from each of the mutant genotypes is not significantly different from one another.
PNA clamping and intercalator detection method (Makito Miyake et al., Biochem Biophys Res Commun., 362:865, 2007) is a method of detecting amplification product by using an intercalator after selectively amplifying only mutant genes while clamping wild-type gene by using a PNA probe. It is not capable of conducting simultaneous multiple detection, and in case the wild-type gene is not completely clamped, false positive results may occur, and thus it is difficult to analyze the results.
The method of using PNA and unlabeled DNA probe (Ji Eun Oh et al., J Mol Diagn., 12:418, 2010) is a method of analyzing the melting curves of an unlabeled DNA probe from a mutant gene by using an intercalator after clamping wild-type gene with a PNA probe. It has problems that it is not capable of specific real-time amplification curve analysis and simultaneous multiple detection and has low sensitivity.